Metabolic disease is caused by a combination of genetic and environmental factors, yet few studies have examined how these factors influence signal transduction, a key mediator of metabolism. Using ...mass spectrometry-based phosphoproteomics, we quantified 23,126 phosphosites in skeletal muscle of five genetically distinct mouse strains in two dietary environments, with and without acute in vivo insulin stimulation. Almost half of the insulin-regulated phosphoproteome was modified by genetic background on an ordinary diet, and high-fat high-sugar feeding affected insulin signalling in a strain-dependent manner. Our data revealed coregulated subnetworks within the insulin signalling pathway, expanding our understanding of the pathway’s organisation. Furthermore, associating diverse signalling responses with insulin-stimulated glucose uptake uncovered regulators of muscle insulin responsiveness, including the regulatory phosphosite S469 on Pfkfb2, a key activator of glycolysis. Finally, we confirmed the role of glycolysis in modulating insulin action in insulin resistance. Our results underscore the significance of genetics in shaping global signalling responses and their adaptability to environmental changes, emphasising the utility of studying biological diversity with phosphoproteomics to discover key regulatory mechanisms of complex traits.
The failure of metabolic tissues to appropriately respond to insulin ("insulin resistance") is an early marker in the pathogenesis of type 2 diabetes. Protein phosphorylation is central to the ...adipocyte insulin response, but how adipocyte signaling networks are dysregulated upon insulin resistance is unknown. Here we employ phosphoproteomics to delineate insulin signal transduction in adipocyte cells and adipose tissue. Across a range of insults causing insulin resistance, we observe a marked rewiring of the insulin signaling network. This includes both attenuated insulin-responsive phosphorylation, and the emergence of phosphorylation uniquely insulin-regulated in insulin resistance. Identifying dysregulated phosphosites common to multiple insults reveals subnetworks containing non-canonical regulators of insulin action, such as MARK2/3, and causal drivers of insulin resistance. The presence of several bona fide GSK3 substrates among these phosphosites led us to establish a pipeline for identifying context-specific kinase substrates, revealing widespread dysregulation of GSK3 signaling. Pharmacological inhibition of GSK3 partially reverses insulin resistance in cells and tissue explants. These data highlight that insulin resistance is a multi-nodal signaling defect that includes dysregulated MARK2/3 and GSK3 activity.
Pancreatic islets are essential for maintaining physiological blood glucose levels, and declining islet function is a hallmark of type 2 diabetes. We employ mass spectrometry-based proteomics to ...systematically analyze islets from 9 genetic or diet-induced mouse models representing a broad cross-section of metabolic health. Quantifying the islet proteome to a depth of >11,500 proteins, this study represents the most detailed analysis of mouse islet proteins to date. Our data highlight that the majority of islet proteins are expressed in all strains and diets, but more than half of the proteins vary in expression levels, principally due to genetics. Associating these varied protein expression levels on an individual animal basis with individual phenotypic measures reveals islet mitochondrial function as a major positive indicator of metabolic health regardless of strain. This compendium of strain-specific and dietary changes to mouse islet proteomes represents a comprehensive resource for basic and translational islet cell biology.
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•Most comprehensive mouse islet proteome library generated to date•Quantification of islet proteomic changes across 6 strains of mice on 2 diets•Islet mitochondrial function revealed as strain-independent regulator of metabolic health
Animal physiology; Diabetology; Proteomics.
Sexually reproducing eukaryotes employ a developmentally regulated cell division program—meiosis—to generate haploid gametes from diploid germ cells. To understand how gametes arise, we generated a ...proteomic census encompassing the entire meiotic program of budding yeast. We found that concerted waves of protein expression and phosphorylation modify nearly all cellular pathways to support meiotic entry, meiotic progression, and gamete morphogenesis. Leveraging this comprehensive resource, we pinpointed dynamic changes in mitochondrial components and showed that phosphorylation of the FoF1-ATP synthase complex is required for efficient gametogenesis. Furthermore, using cryoET as an orthogonal approach to visualize mitochondria, we uncovered highly ordered filament arrays of Ald4ALDH2, a conserved aldehyde dehydrogenase that is highly expressed and phosphorylated during meiosis. Notably, phosphorylation-resistant mutants failed to accumulate filaments, suggesting that phosphorylation regulates context-specific Ald4ALDH2 polymerization. Overall, this proteomic census constitutes a broad resource to guide the exploration of the unique sequence of events underpinning gametogenesis.
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•Coordinated waves of protein expression and phosphorylation drive gametogenesis•Sequential kinase activities shape the phosphorylation landscape throughout meiosis•Phosphorylation of the FoF1-ATP synthase complex is required for efficient meiosis•Phosphorylation regulates meiotic assembly of mitochondrial Ald4ALDH2 filaments
Wettstein et al. employ integrated proteomics and cryoET to study the process of meiosis in yeast and report that sequential waves of protein phosphorylation rewire the proteome during gametogenesis. Phosphorylation of the FoF1-ATP synthase complex is required for efficient meiosis, and phosphorylation promotes the meiotic assembly of mitochondrial Ald4ALDH2 filaments.
Metabolic disease is caused by a combination of genetic and environmental factors, yet few studies have examined how these factors influence signal transduction, a key mediator of metabolism. Using ...mass spectrometry-based phosphoproteomics, we quantified 23,126 phosphosites in skeletal muscle of five genetically distinct mouse strains in two dietary environments, with and without acute in vivo insulin stimulation. Almost half of the insulin-regulated phosphoproteome was modified by genetic background on an ordinary diet, and high-fat high-sugar feeding affected insulin signalling in a strain-dependent manner. Our data revealed coregulated subnetworks within the insulin signalling pathway, expanding our understanding of the pathway's organisation. Furthermore, associating diverse signalling responses with insulin-stimulated glucose uptake uncovered regulators of muscle insulin responsiveness, including the regulatory phosphosite S469 on Pfkfb2, a key activator of glycolysis. Finally, we confirmed the role of glycolysis in modulating insulin action in insulin resistance. Our results underscore the significance of genetics in shaping global signalling responses and their adaptability to environmental changes, emphasising the utility of studying biological diversity with phosphoproteomics to discover key regulatory mechanisms of complex traits.
Insulin-induced GLUT4 translocation to the plasma membrane in muscle and adipocytes is crucial for whole-body glucose homeostasis. Currently, GLUT4 trafficking assays rely on overexpression of tagged ...GLUT4. Here we describe a high-content imaging platform for studying endogenous GLUT4 translocation in intact adipocytes. This method enables high fidelity analysis of GLUT4 responses to specific perturbations, multiplexing of other trafficking proteins and other features including lipid droplet morphology. Using this multiplexed approach we showed that Vps45 and Rab14 are selective regulators of GLUT4, but
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knockdown affected both GLUT4 and TfR translocation. Thus, GLUT4 and TfR translocation machinery likely have some overlap upon insulin-stimulation. In addition, we identified Kif13A, a Rab10 binding molecular motor, as a novel regulator of GLUT4 traffic. Finally, comparison of endogenous to overexpressed GLUT4 highlights that the endogenous GLUT4 methodology has an enhanced sensitivity to genetic perturbations and emphasises the advantage of studying endogenous protein trafficking for drug discovery and genetic analysis of insulin action in relevant cell types.
BRCA1-mutated breast cancer is primarily driven by DNA copy-number alterations (CNAs) containing large numbers of candidate driver genes. Validation of these candidates requires novel approaches for ...high-throughput in vivo perturbation of gene function. Here we develop genetically engineered mouse models (GEMMs) of BRCA1-deficient breast cancer that permit rapid introduction of putative drivers by either retargeting of GEMM-derived embryonic stem cells, lentivirus-mediated somatic overexpression or in situ CRISPR/Cas9-mediated gene disruption. We use these approaches to validate Myc, Met, Pten and Rb1 as bona fide drivers in BRCA1-associated mammary tumorigenesis. Iterative mouse modeling and comparative oncogenomics analysis show that MYC-overexpression strongly reshapes the CNA landscape of BRCA1-deficient mammary tumors and identify MCL1 as a collaborating driver in these tumors. Moreover, MCL1 inhibition potentiates the in vivo efficacy of PARP inhibition (PARPi), underscoring the therapeutic potential of this combination for treatment of BRCA1-mutated cancer patients with poor response to PARPi monotherapy.
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